Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute in complex fashion to cell migration and signaling. We are addressing the following questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. Spatial regulation by the topography of the local microenvironment can alter a wide variety of cellular functions. In order to analyze in depth the roles of topological organization of extracellular matrix molecules, we developed a procedure termed micro-photoablation. Detailed protocols for this technique have been developed and standardized for use by other laboratories. This new methodology permits the generation of any type of pattern of matrix proteins. For example, it can generate micron-wide patterned lines that serve as efficient paths of cell migration, which closely mimics cell migration along three-dimensional (3D) cell-derived fibrils. We term this process of cell migration along a narrow line of matrix proteins "1D migration." We previously showed that many aspects of 3D cell migration in a 3D fibrillar matrix, including morphology, migration, cytoskeletal organization, and responses to matrix density, are reproduced much more effectively by this process of 1D migration compared to the mode of migration on traditional flat, 2D surfaces routinely used for cell culture. Cells can develop intracellular tension using cellular actin and myosin, and they can sense tension in their microenvironment in the process of cellular mechanotransduction. A particularly striking discrepancy was identified in cellular responses to inhibitors of contractility during cell migration under 1D and 3D versus 2D conditions. Treatment of fibroblasts with inhibitors of actomyosin contractility on 2D fibronectin-coated surfaces leads to slight increases in rates of cell migration, whereas the same treatments of cells plated on 1D (fibronectin-coated) or 3D fibrillar cell-derived matrix substantially inhibits migration by greater than two-fold. Based on these observations, together with our finding that adhesions to the underlying substratum in 1D form a unique, lengthy adhesion structure unlike that found on 2D surfaces, we are testing whether changing the topography and physical structure of cell-ECM adhesions affects the basic morphological and biochemical mechanisms proposed to mediate cell migration. In order to quantify the dynamics of proteins potentially comprising a clutch-like mechanism implicated in cell-matrix interactions, we are currently analyzing fluorescence recovery after photobleaching (FRAP) of GFP-linked fusion proteins together with other live-cell imaging techniques (spinning disk and TIRF microscopy) to track the dynamics of the proteins involved in the postulated molecular clutch to determine how 1D ECM enhances cell migration. More generally, we feel that studying cells migrating in 1D will provide a powerful new tool for analyzing the molecular mechanisms of cell migration, because the components of the molecular machinery are arrayed linearly along the length of a steadily migrating cell that remains oriented in a single direction. Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. We and others recently published studies on the roles of the major myosin II genes, myosin IIA and IIB. We found that myosin IIA plays central roles in fibroblast and embryonic stem cell contractility, actin cytoskeletal organization, and organization of cell-matrix adhesions. We are now directly comparing the roles of myosin IIA and IIB isoforms in 1D, 2D, and 3D cell migration systems. We had also previously identified strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. We are exploring the mechanisms of this cross-talk between these two major cytoskeletal systems in 2D and 3D systems. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosins and microtubules. All of these processes need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.